The goal of this research is to identify,clone,and characterize TSGs located on chromosomes 3p and 8p that are involved in the origin or development of major human malignancies: carcinomas of the lung, breast,kidney, and prostate. Our major accomplishments this year are: (1) assembly of the complete sequence of the VHL gene (GenBank #AFO10238);(2) identification of pVHL target genes by differential display technology; to date 25% of the cell transcibed genes were analyzed and six down regulated genes identified, namely,NOTCH 2,that specifies cell fate determination,RACK 1 an intracellular receptor for PKC, CA9 and10 (members of the carbonic anhydrase superfamily), and two new unknown genes. (3) analysis of the methylation of the VHL promoter in renal carcinoma:monochromosome gene transfer, cell fusion, and VHL gene transfections showed that the meth+ phenotype is dominant. (4)The 3p21.3 TSG:the 370kb critical region defined and sequenced last year is now narrowed to 120kb by a newly discovered overlapping deletion.Eight new genes reside in this region,including a new alpha2- delta subunit of the L-type calcium channel that appears to elicit autoantibodies in LEMS (Lambert - Eaton myastenic syndrom) associated with SCLC. These genes are being analyzed for mutations, expression,imprinting, and the ability to suppress growth of target cancer cells. (5)The 3p12 TSG: a candidate gene that is a new member of the cell adhesion molecule superfamily (CAMs) and showed homology to the TSG DCC is being analyzed (6)The 3p25-p26 TSG:using in silico location cloning a new member of CAMs,CALL,was isolated and analysed (GenBank #AFO02246).(7)The 8p22 TSG: products of two classical TSGs, NF1 and TSC2 are large GTPase activating proteins (GAPs) for RAS and RAP1 respectively.To discover other GAPs we searched NCBI databases for ESTs representing such genes in TSGs regions defined by LOH on chromosomes 8p and 1p.We have now isolated and characterized the human ortholog of the rat p122RhoGAP, which is a dual regulator of Rho and PLC delta. This gene is localized by us to 8p22 (GenBank # AFO26219) and is a candidate TSG for lung, breast, and prostate cancers.